Plasmids are self-replicating DNA molecules that exist naturally in bacteria, archaea and some unicellular eukaryotes, such as yeast. In recent years they have become essential to the biotechnology industry for the expression of recombinant protein genes and as DNA therapeutics and vaccines. For such applications plasmids encoding genes of interest are generally modified and replicated in a bacterial host cell such as Escherichia coli. Plasmids often encode an antibiotic resistance gene to enable antibiotic selection to be used to identify the cells which contain the plasmid following transformation, with the selective antibiotic added to the growth medium to kill cells that have lost the plasmid.
However, there are several disadvantages to using antibiotics for plasmid selection and maintenance. Firstly, the constitutive expression of the antibiotic resistance gene in the host cell produces a metabolic burden on the cell that reduces viability and increases the frequency of plasmid loss. Secondly, the antibiotics represent an additional contaminant in manufacture, and selection pressure is reduced by antibiotic degradation during fermentation. Thirdly, for DNA therapeutics and vaccines, the use of antibiotic resistance genes carries the risk of transfer to pathogens in the environment, leading to antibiotic resistant pathogenic strains. This is an acute risk when live bacterial strains are used as vectors for gene delivery to a patient. There is therefore a requirement to develop a mechanism of plasmid selection without the use of antibiotic resistance genes.
Alternative technologies have been developed that require an expressed selectable marker gene, such as a functional copy of an essential gene that complements a deleted copy on the host chromosome. The thymidylate synthase gene thyA (McNeil et al., 2000, Appl. Environ. Microbiol., 66: 1216-1219) or asd gene involved in diampinopimelic acid synthesis (Degryse 1991, Mol. Gen. Genet. 227: 49-51) have been used as the selective genes on plasmids in cells where the chromosomal genes are non-functional. Both this approach and antibiotic selection share the same important drawback: the presence and expression of a selectable marker gene that results in a significant metabolic burden to the cell and makes plasmid loss selectively advantageous (Bentley et al. 1990, Biotechnol. Bioeng. 35: 668-681).
Two further technologies have been developed which circumvent the requirement for selectable marker gene expression, and therefore lead to a reduced metabolic burden on the cell. ORT (Operator-Repressor Titration) utilises a modified bacterial cell where an essential chromosomal gene is placed under the control of an inducible promoter. A repressor protein binds to operator sequences adjacent to the promoter to prevent expression of the essential gene, thus causing the cell to die unless an inducer is present. When an ORT bacterial cell is transformed with a multi-copy plasmid containing the operator sequence, the repressor is titrated by the plasmid and the expression of the essential gene is enabled, thus allowing cell growth and therefore plasmid selection and maintenance (Cranenburgh et al. 2001. Nucleic Acids Res. 29: e26).
The other selectable marker gene-free system, oriSELECT, utilises the pMB1 origin of replication that is found on the majority of plasmids used in molecular genetics research and development. The pMB1 on naturally produces an antisense RNA to regulate its copy number, and oriSELECT cells are modified such that this RNA interacts with the mRNA of a corresponding sense sequence engineered in a gene fusion with either a repressor regulating an essential gene, or a toxin gene, such that the presence of the plasmid is required for cell survival (Cranenburgh 2005, WO06/003412).
The disadvantage of both of these selectable marker gene-free expression systems is that the chromosomes of the microbial cells need to be genetically modified. This can be technically challenging in many species, and even in species that are readily amenable to genetic manipulation it is time-consuming and laborious. There therefore remains a need to develop a plasmid selection system would be free from selectable marker genes and which would not require genetic modification of the host cell.